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Abstract:

The present invention is a dentinal drug delivery composition composed of
cationic and/or neutral porous particles containing an effective amount
of a therapeutic agent, a method for using the dentinal drug delivery to
provide a dental treatment, and a method for identifying
anti-inflammatory agents capable of diffusing through dentin.

Claims:

1. A dentinal drug delivery composition comprising cationic and/or
neutral porous particles containing an effective amount of a therapeutic
agent, said particles in admixture with a carrier suitable for attachment
of the particles to the dentin.

3. The composition of claim 1, wherein the therapeutic agent is an
anti-inflammatory drug that controls pulpal inflammation.

4. The composition of claim 3, wherein the anti-inflammatory drug is
2-amino-3-benzoylbenzeneacetamide, acetylsalicyclic acid, ibuprofen, or
an ω-3 fatty acid.

5. The composition of claim 1, wherein the therapeutic agent is an
antibiotic.

6. The composition of claim 1, wherein the therapeutic agent is an
analgesic.

7. A method for providing dental treatment comprising administering to a
subject in need thereof, the dentinal drug delivery composition of claim
1, thereby providing a dental treatment to the subject.

8. A method for identifying an anti-inflammatory agent for decreasing
inflammation in dental pulp comprising (a) contacting a coronal surface
of an isolated dentin sample with a test agent, (b) incubating the test
agent and dentin sample so that diffusate is formed, (c) contacting an
isolated dental pulp cell with the diffusate, and (d) determining whether
the diffusate comprises an amount of test agent capable of decreasing the
expression or activity of at least one inflammatory mediator of the
dental pulp cell thereby identifying an anti-inflammatory agent for
decreasing inflammation in dental pulp.

9. The method of claim 8, wherein the test agent is a non-steroidal
anti-inflammatory drug.

10. The method of claim 8, wherein the test agent is an ω-3 fatty
acid.

11. An anti-inflammatory agent identified by the method of claim 8.

Description:

INTRODUCTION

[0001] This application is a continuation-in-part application of U.S.
patent application Ser. No. 13/511,008, filed May 29, 2012, which is a
U.S. National Stage Application of PCT/US2010/057718 filed Nov. 23, 2010
and claims benefit of priority to U.S. Provisional Application Ser. No.
61/263,510 filed Nov. 23, 2009, the contents of each of which are
incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] Dentin is a biological composite composed of both inorganic and
organic components. The chemical and physical characteristics of
surgically exposed dentin determine the ability of therapeutic and
restorative materials to adhere to these surfaces. In vital-permeable
dentin there is a continuous outward-flow of dentin fluid. Materials
applied to the dentin surface may be able to penetrate the dentinal
tubules but fail to be retained if they are incompatible with this fluid
or are dislodged by its flow. When dentin surfaces are demineralized by
acid etching, a layer of collagen fibers are exposed. Hydrophilic dentin
bonding agents can interpenetrate this hydrated collagen layer resulting
in a strong bond between dentin and restorative materials (Nakabayashi
(1992) Proc. Finn. Dent. Soc. 88 Suppl 1:321-9). Hydrogen bonding and van
der Waals forces have been found to be responsible for the interaction
between collagen and several types of dentin primers.
Hydroxyethylmethacrylate (HEMA) can associate with collagen through sites
on the collagen molecule that act as ligands for this polymer
(Vaidyanathan, et al. (2003) J. Adhes. Dent. 5:7-17). In addition, agents
such as gluteraldyhyde can covalently link primers to the dentin
collagen.

[0003] Electrostatic interactions may help anchor restorative and other
therapeutic materials to dentin. Many biological surfaces have fixed
negative charges due to the presence of proteins, and other
macromolecules, containing carboxylated, sulfonated or phosphorylated
functional groups that are ionized at physiological pH. For example,
clean hair has a net anionic surface charge (Ungewiss, et al. (2005)
Anal. Bioanal. Chem. 381:1401-7). Several types of cationic polymers
containing quaternary nitrogen functional groups adhere to hair and are
used as conditioners. Cationic antimicrobial agents such as chlorhexidine
are substantive to many oral surfaces including dentin (Rosenthal, et al.
(2004) Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 98:488-92).
As is the case with other biological substrates, utilization of
electrostatic forces should help attach material to dentin surfaces. The
mineral phase of dentin provides other means by which organic molecules
can bind to this tissue. Organic acids can both demineralize and bind to
dentin. Many low molecular weight organic acids such as citric and lactic
acid form soluble calcium salts. These acids demineralize dentin
(Yoshida, et al. (2001) J. Dent. Res. 80:1565-9). Higher molecular weight
polyalkenoic acids bind exposed calcium ions on the dentin surface
forming tenacious gels that resist removal. Calcium ions in the fluid
layer adjacent to the dentin surface can also contribute to adhesion by
forming ionic cross-bridges between polymers containing organic acid
groups and anionic macromolecules in dentin (Hannig & Hannig (2009) Clin.
Oral Investig. 13:123-39). Since dentin possesses a variety of chemical
functionalities, different binding mechanisms can be employed to attach
materials to the dentin surface.

[0004] Dentin has a markedly inhomogeneous structure. The dentinal tubules
become wider and are more numerous in deep dentin, close to the dental
pulp. Since each tubule has a thin sheath of organic material (the lamina
limitans) lining the lumen of each tubule, the density of organic
material would be expected to be greater in deep as opposed to shallow
dentin (Thomas (1984) J. Dent. Res. 63:1064-6). In addition to collagen,
the dentin contains a variety of other proteins such as dentin
phosphosphoryn, which are highly phosphorylated and have low pKa values
(Butler (1995) Connect. Tissue Res. 33:59-65). These proteins are
believed to play a role in dentin mineralization and when decomplexed
from calcium by etching would become anionic. In addition proteoglycans
are present particularly in the predentin (Waddington, et al. (2003)
Matrix Biol. 22:153-61).

[0005] The chemical characteristics of dentin's intratubular organic
material can affect the diffusion of solutes through dentin. Most low
molecular weight anionic materials such as pertechnetate and iodine ions
diffuse readily through dentin (Pashley, et al. (1977) J. Dent. Res.
56:83-8). In contrast, chlorhexidine, a cationic agent, has a lower
dentin permeability coefficient than is predicted on the basis of its
molecular weight (Pashley & Livingston (1978) Arch. Oral Biol. 23:391-5).
A basic polypeptide (parathyroid hormone; Pichette, et al. (2000) J.
Chromatogr. A 890:127-33) was found to be unable to diffuse through
dentin unless it was absorbed onto albumen, an anionic protein (Pashley
(1988) Int. Endod. J. 21:143-54). The results of these diffusion
experiments indicate that dentin binds cationic molecules hindering their
transdentinal diffusion. This behavior is consistent with the view that
the etched-dentin surface and tubule walls have fixed anionic charges and
provide binding sites for cationic materials.

[0006] Alternatively, it has been proposed that the dentinal tubules are
filled with a cationic gel of unspecified composition (Linden, et al.
(1995) Arch. Oral Biol. 40:991-1004). This gel was observed using
scanning-probe microscopy. In in vitro experiments measuring dentin flow,
treatment of dentin slices with proteolytic enzymes resulted in a large
increase in flow, indicating that proteins including collagen partially
reduced the effective diameter of the dentinal tubules (Linden, et al.
(1995) supra). Transdentinal diffusion of negatively charged myoglobin
was restricted as compared to neutral myoglobin even though both proteins
had the same ionic radius. These observations lead these investigators to
conclude that the intertubular gel had fixed positive charges.

[0008] The present invention is a dentinal drug delivery composition
composed of cationic and/or neutral porous particles containing an
effective amount of a therapeutic agent, wherein the particles are in
admixture with a carrier suitable for attachment of the particles to the
dentin. In one embodiment, the porous particle is composed of silicon or
latex. In other embodiments, the therapeutic agent is an
anti-inflammatory drug that controls pulpal inflammation; an antibiotic;
or an analgesic. A method for providing dental treatment with the
composition of this invention is also provided.

[0009] This invention also provides a method for identifying an
anti-inflammatory agent for decreasing inflammation in dental pulp. This
method includes the steps of contacting a coronal surface of an isolated
dentin sample with a test agent, incubating the test agent and dentin
sample so that diffusate is formed, contacting an isolated dental pulp
cell with the diffusate, and determining whether the diffusate has a
sufficient amount of test agent to decrease the expression or activity of
at least one inflammatory mediator of the dental pulp cell thereby
identifying an anti-inflammatory agent for decreasing inflammation in
dental pulp. In some embodiments, the test agent is a non-steroidal
anti-inflammatory drug or an ω-3 fatty acid.

[0013] Within each vital tooth there is a small organ rich in nerves and
blood vesicles called the dental pulp. Disease such as caries or trauma
can cause inflammation in this delicate tissue. Pulpal inflammation often
results in the severe pain commonly referred to as toothache. Since the
pulp is enclosed within the dental hard tissues, the circulatory events
occurring during severe inflammatory processes frequently result in loss
of blood supply and tissue death. Even mild inflammatory responses such
as those occurring following the placement of a routine dental
restoration can result in post-operative sensitivity. Conventional
treatment of pulp inflammation is limited. Patients with severe pulp
inflammation require removal of the offending tooth or removal of the
pulp (endodontic therapy, also known as root canal treatment) with
conservation of the tooth. Endodontic therapy is expensive and time
consuming; hence by increasing the ability to manage pulpal inflammation
pharmacologically, dental care is improved.

[0014] It has now been found that porous particles are of use in
delivering, e.g., anti-infective or anti-inflammatory drugs as well as
biological agents such as growth and biological response modifying
factors to diseased and injured dental pulps in order to improve
treatment outcomes and allow dental procedures to be delivered in a more
predictable and cost effective manner. Once attached to the dentin, these
particles can release drug that reaches the site of action by diffusing
through the fluid-filled tubules that are a prominent component of the
dentin structure. The drug carrying particles can be nondegradable and
form part of the interface between the dentin and the restorative
material. Procedures for fabricating porous silica particles over a range
of clinically useful particle diameters have been described in the
literature and the materials are commercially available.

[0015] Accordingly, the present invention features a dentinal drug
delivery composition composed of cationic or neutrally charged porous
microparticles containing an effective amount of a therapeutic agent,
wherein said particles are in admixture with a carrier suitable for
attachment of the microparticles to the dentin. As used herein, the terms
"microspheres," "particles" and "microparticles" are essentially
synonymous. These microspheres, which are generally spherical in shape,
are typically sized to have nominal diameters in the range of from about
0.1 micron to about 1 micron. In particular embodiments, the average
diameter of the microspheres of the invention are about 0.5 micron.

[0016] By "porous" is generally meant a porosity of at least about 50%,
and preferably a porosity of from about 50% to about 65%. The degree of
porosity refers to the total pore volume within the solid support, e.g.,
silica particle. Porosity increases with increasing pore volume.

[0017] The porous microspheres of the present invention can be prepared by
any conventional method. In particular embodiments, the microsphere of
the invention is composed of silica or latex. By way of illustration, the
silica particles can be prepared by spray drying silica solutions made by
the controlled hydrolysis of tetraethyl-o-silicate or similar organic
silicon compounds. This method allows the formation of highly purified
porous silica microspheres at a relatively low cost and with highly
controlled properties. The silica support can be made with different
particle sizes and different pore sizes to, e.g., accommodate the
therapeutic agent being delivered.

[0018] More specifically, appropriate silica sols can be prepared by the
hydrolysis of organic silicates in the manner described by Stober, et al.
(1968) J. Colloid and Interface Science 26:62-69. This approach is known
to make silica sols with a very high purity and with a narrow sol
particle size distribution. The particle size of the sol prepared in this
manner determines the pore size of the porous silica microspheres
ultimately made from these sols, with the average pore size being about
one-half the average diameter of the silica sol microparticles.

[0019] Porous silica microspheres can then be made from these aqueous
colloidal silica sols by using well known spray-drying equipment and
methods (Masters, Spray Drying Handbook, 5th ed. Longman Scientific
and Technical, New York (1991)). In some embodiments, the silica
solutions are first be flocculated or partially pre-gelled by using a
process such as described in Iler, The Chemistry of Silica, Chapter 4,
John Wiley, New York (1979), to produce microspheres with a porosity that
is higher than that available by the direct spray drying of silica sols.
The concentration of the silica solution, the type and rate of the
spray-drying nebulization (for example, two-fluid nozzle or spinning
disk), the drying temperature, the rate of heated air supply, and the
like, are all adjusted to produce the porous silica microspheres of the
desired size and size distribution.

[0020] In particular embodiments, porous silica particles contain
functional groups on the surface thereof that impart a neutral or
positive charge. A cationic charge on the surface of the instant
particles can be incorporated by conventional methods using positively
charged groups selected from the group of primary amine, secondary amine,
tertiary amine, quaternary ammonium salts, amidines, pyridinium salts and
mixtures thereof. Exemplary groups include amine and amidine groups.

[0022] The particle sizing may be accomplished by a number of well-known
methods, such as sieving, air classification, and liquid elutriation.
Sieving is the simplest and least costly method. However, this method
produces products that have the greatest concentration of fine particles,
because of the tendency of fines to adhere to larger particles and
therefore not be properly fractionated. Air classification with a
relatively expensive machine is a convenient method that permits a high
throughput of desired particles to be fractioned accurately in a narrow
particle size distribution.

[0023] It is further contemplated that the microspheres of the invention
can be coated with various inorganic and organic constituents in order to
impart the particles with affinity for surgically prepared and
pathologically altered dentin and/or the ability to hold and release
various pharmacological agents.

[0024] In contrast to the absorption of a therapeutic substance on the
surface, the instant microspheres are porous and find application in
holding and releasing a variety of therapeutic agents used in the field
of dentistry. Examples of topically active drugs that could be released
from a microsphere of the invention include, but are not limited to,
analgesic agents; anti-inflammatory agents such as
2-amino-3-benzoylbenzeneacetamide (NEPAFENAC), acetylsalicyclic acid
(aspirin), ibuprofen, diclofenac, difusinol, indomethacin, an ω-3
fatty acid or other monounsaturated or polyunsaturated fatty acid (e.g.,
oleic acid or docosahexaenoic (DHA)); anti-microbial agents such as
chlorhexidine; and fluoroquinolone antibiotics such as ofloxacin.

[0025] For use in vivo, particles of the present invention can be prepared
as pharmaceutical compositions, wherein the particles are in admixture
with a carrier suitable for attachment of the particles to the dentin. In
this respect, the carrier selected does not change or mask the charge of
the particle. Suitable carriers include, but are not limited to water,
saline or other conventional carrier used in dental applications.

[0026] The ability of the instant porous microspheres to adhere to dentin
and release drugs can be examined via a variety of in vitro methods. For
example, the ability of a porous particle to adhere to dentin surfaces
can be assessed using an in vitro assay as described herein. Part of the
intertubular dentin matrix and the walls of the tubules are composed of
organic macromolecules. In this respect, adherence of dyes and
particulate material to dentin can be readily assessed by
photomicrographic means. As shown here, untreated dentin stained with
toluidine blue has a deep purple-blue color that is indistinguishable
from the dye solution itself. The pattern of dentin's staining with
toluidine blue has been described in demineralized, dehydrated tissue
(Major (1966) Arch. Oral Biol. 11:1293-305). The intratubular dentin very
close to the pulp stains intensely. Moving away from the pulp, a poorly
stained region is encountered, then a zone where material inside the
tubule lumen is stained. The superficial dentin is observed to be lightly
stained. The biochemical nature of the tissue determines the color
resulting from toluidine blue staining. Large amounts of acidic molecules
in tissue effect the orientation and spacing of dye molecules resulting
in a color shift to red. This phenomenon is called metachromasia and was
observed in histological sections of developing mouse dentin
(Ravindranath & Basilrose (2005) Acta Histochem. 107:43-56). Treatment of
the tissue sections with enzymes that cleave acidic functional groups off
of proteins reduces the degree to which metachromasia is observed,
indicating that this type of staining is very sensitive to the chemical
composition of tissue.

[0027] The heavy orthochromatic staining observed herein with toluidine
blue indicates that dentin is rich in anionic molecules. In the dentin
sections cut perpendicular to the long axis of the tooth, dentin
overlying the pulp horn area was observed to be particularly deeply
stained. This is the region of dentin with the widest tubules, the
highest permeability (Pashley, et al. (1987) Arch. Oral Biol. 32:519-23)
and the greatest density of intratubular organic material. In vivo, the
outward flow of dentin fluid limits the diffusion driven penetration of
dyes through the tubules. The experiments in this study were performed
without the application of simulated pulp pressure, since the goal was to
use dye staining as a means to examine the binding properties of dentin.
Since extracted teeth were used in this study, organic material from the
dental pulp and remnants of the odontoblasts may have leaked into the
tubules and increased the intensity of the staining. The enamel portions
of the tooth slices stained lightly with toluidine blue indicating that
the dye has some affinity for hydroxyapatite. Similar light staining can
also be observed when squares made of sintered hydroxyapatite are treated
with toluidine blue.

[0028] In marked contrast to untreated dentin, dentin that was pretreated
with the cationic polymer solution was only faintly stained. Chroma meter
readings indicated that unstained dentin has a high brightness value and
a positive reading on the yellow-blue scale indicating yellow color.
Following toluidine blue staining, the brightness value significantly
dropped and a significant shift in the yellow blue parameter occurred to
a negative value, indicating that the dentin color had become blue.
Brightness values from dentin surfaces treated first with the cationic
polymer than stained with toluidine blue were not significantly different
from those of unstained dentin. The yellow-blue scale reading of dentin
that was polymer treated then toluidine blue stained was significantly
lower than that of unstained dentin, indicating that some shift in dentin
color from yellow to blue occurred. This pale blue staining with more
intense staining of the pulp horn region was evident in the cationic
polymer-treated stained dentin. These results showed that treatment of
the dentin with the cationic polymer reduced, but did not entirely block
toluidine blue staining.

[0029] The ability of porous particles, found to have affinity for dentin
in the experiments described above, can be examined in conventional
release assays as described in the literature. Moreover, the
transdentinal diffusion of drug released from particles can be measured.
Due to the chemical properties of the dentin, the transdentinal diffusion
of certain solutes is restricted. Dentin sections can be placed into a
dentin diffusion cell. Loaded particles can be applied to the outer
dentin surface while the fluid on the inner (pulpal) side of the disk is
withdrawn at regular intervals and analyzed for the presence of the drug.
This experiment allows for the evaluation of drug release and delivery
kinetics.

[0030] The findings herein have significant clinical applications.
Therefore, the instant invention also includes a method for providing
dental treatment by administering to a subject in need thereof, the
dentinal drug delivery composition of the invention. The observation that
charged acidic particulates and cationic materials adhere to etched
dentin indicates that these types of agents can be incorporated into
restorative materials or agents used to topically desensitize teeth.
Charged particulates can also be used as dentin drug delivery
compositions, where drugs can be loaded into hollow or porous
microspheres that can release the drug into the dentinal fluid in a
diffusion controlled manner over a period of time. Following trauma or
the excavation of deep decay, pulpal inflammation can cause pain and
eventual loss of tooth vitality. New insights into the peripheral
mechanisms of dental pain can lead to the development of new analgesic
drugs that target peripheral intradental nerve endings. When dentin
(particularly deep dentin) is etched, fluid flows in an outward direction
through the patent tubules. This outward flow opposes the inward
diffusion of solutes, including drugs, through the dentin. Following
placement of a bonded restoration over the exposed dentin the outward
flow ceases. Placing the instant drug delivery composition between the
dentin and the restorative material would allow the drug to diffuse into
the dentinal fluid without the opposing influence of this outward fluid
flow. Drug delivery compositions that have an affinity for deep dentin
can be applied as part of the restorative treatment releasing drug into
the dentinal fluid while serving as the interface between the dentin and
the bulk of the restoration (FIG. 2).

[0031] The use of hollow or porous silica particles as drug carriers is
illustrated in FIG. 3. Following the removal of deep tooth decay, the
dentist would place the particulate containing drug delivery composition
onto the exposed dentin. The drug carrier would then be covered by the
tooth restorative material. Once in contact with the dentin, drug would
diffuse out of the particle and through the dentinal fluid to its site of
action in the dental pulp. The tooth dentin is composed of a partially
mineralized matrix containing collagen and other proteins. Fluid filled
tubules, 0.5-2.5 μm in diameter transverse the thickness of the
dentin. The instant delivery composition can be designed so that it
adheres to the dentin and releases the pharmacological agent, which
subsequently diffuses through the fluid filled tubules to the neural,
vascular or other sites of action in the superficial pulp tissue. Since
the acute phase of pulp inflammation peaks about one week after injury,
drug delivery for that period of time would be effective in modulating
the inflammatory response. Based on in vitro experiments utilizing 0.5
μm latex spheres, as described herein, the surface characteristics of
particulate materials that adhere to surgically prepared dentin surfaces
have now been defined. Due to the heterogeneous nature of the dentin,
beads with a variety of surface chemistries were found to have affinity
for this tissue.

[0032] For drugs to be effective, they must be capable of diffusing
through the dentinal tubules. Dentin acts a permselective membrane where
electrostatics, aqueous solubility and molecular weight mediate diffusion
of a molecule through the dentin. Therefore, to identify agents that can
diffuse through a dentin barrier in sufficient amounts and act on dental
pulp cells, this invention also provides an in vitro cell-based method
for assessing drug diffusion through dentin and activity against dental
pulp cells. Determining whether agents can diffuse through dentin is
critical in determining which agents can be useful in a dentin
drug-delivery system and provide therapeutics to the dental pulp
following invasive dental processes.

[0033] Agents that can be screened using this method of the invention
include, but are not limited to, analgesic agents; anti-inflammatory
agents such as 2-amino-3-benzoylbenzeneacetamide (NEPAFENAC),
acetylsalicyclic acid (aspirin), ibuprofen, diclofenac, difusinol,
indomethacin, an ω-3 fatty acid or other monounsaturated or
polyunsaturated fatty acid (e.g., oleic acid or docosahexaenoic (DHA));
anti-microbial agents; and antibiotics. Moreover, combinations of drugs
and dosage forms can be evaluated using the method of this invention.
Synergies between anti-inflammatory and water-soluble analogs of
ω-3 fatty acids can also be examined.

[0034] This screening method of the invention includes the steps of
contacting a coronal surface of an isolated dentin sample with a test
agent, incubating the test agent and dentin sample so that diffusate is
formed on the pulpal side of the dentin sample, contacting an isolated
dental pulp cell with the diffusate, and determining whether the
diffusate contains a sufficient amount of test agent to act on a cell of
the dental pulp. In one embodiment, it is determined whether the
diffusate contains a sufficient amount of test agent to inhibit or
decrease the expression or activity of at least one inflammatory mediator
of the dental pulp cell (e.g., nociceptive nerve endings, blood vesicle
cells or inflammatory cells).

[0035] The dentins sample can be isolated by conventional methods and used
in combination with a commercially available two-chamber diffusion cell,
wherein the partition separating the two chambers holds the dentin sample
(e.g., a dentin disk) in an orientation so that the dentin tubules
provide the only connection between the two chambers. See, e.g., Hanks,
et al. (1993) J. Dent. Res. 72:931-938; and Puapichartdumrong, et al.
(2003) Internatl. Endodont. J. 36:674-81. The diffusant (i.e., test
agent) is loaded into one cell (the donor compartment) then following one
or more time intervals, the presence and concentration of the diffusant
is ascertained in the second cell (receptor compartment). Test agent
found in the receptor compartment during the course of the experiment
reached that chamber by diffusing through the dentin barrier.

[0036] After the one or more time intervals (e.g., one or more minutes,
hours, or days), the diffusate is collected and tested for activity
against an isolated dental pulp cell or isolated population of dental
pulp cells. An isolated pulpal cell or population of dental pulp cells
refers to cells that are removed from their native environment (i.e., the
dental pulp). The isolated cells can be cultured primary cells or
cultured cell line and include, but are not limited to fibroblasts,
odontoblasts, histiocytes, macrophage, granulocytes, mast cells or plasma
cells. Exemplary cell lines include, but are not limited to the murine
macrophage cell line, RAW 264.7; immortalized mouse odontoblast cell
line, MDPC-23; and the murine fibroblast cell line, 3T3.

[0037] As indicated, in some embodiments it is determined whether the
diffusate contains a sufficient amount of test agent to inhibit or
decrease the expression or activity of at least one inflammatory mediator
of the dental pulp cell. In accordance with this embodiment, the isolated
pulpal cell or population of dental pulp cells can be challenged with an
inflammation-provoking stimulus (e.g., Lipopolysaccharide (LPS),
N-formyl-methionyl-leucyl-phenylalanine, or phorbol myristate acetate)
either before or after being contacted with the diffusate, so that the
effect of the drug diffusing through the dentin barrier on inflammatory
mediator production is assessed.

[0038] Prostaglandins such as PGE2 and nitric oxide (NO) are inflammatory
mediators that are generated in injured tissue and influence vascular
tissues, nociceptive nerve endings and immune cells. For example,
exposure of immune and other cells to bacterial toxins such as LPS evokes
production of PGE2 and NO. Other known mediators of inflammation include,
but are not limited to, bradykinin, histamine, leukotrienes (Bd,
Cd, Dd, Ed) IL-8, and neutrophil and macrophage lysosomal
enzymes. The effect of test agents on the expression or activity of one
or more of the above-referenced inflammatory mediators can be determined
by conventional methods. For example, mRNA expression can be determined
by dot blot, northern blot, or reverse transcriptase (RT)-PCR; protein
expression can be determined by western blot or ELISA analysis; and NO
production can be determined using a Griess reaction assay (e.g.,
EMSNOTOT kit) or ozone-based chemiluminescence assay.

[0039] A reduction or decrease in the level or activity of one or more
inflammatory mediators as compared to a control cell or population of
cells, e.g., cells not contacted with the test agent, indicates that a
sufficient amount of the test agent diffused through the dentin to act on
the isolated cell or population of cells of the dental pulp. By measuring
the responses of cells to a series of known drug doses, a dose response
curve can be generated. The dose response curve can be used to calculate
the drug concentration generated in the second cell (receptor
compartment) in individual diffusion experiments. Data collected in these
experiments can be used to determine the transdentinal flux of individual
drugs using Fick's equation:

Flux=-PA(cB-cA)

where: A is the area of the dentin disk available for diffusion; (cB-cA)
is the difference in concentration between the second (cB) and first (cA)
chambers, and P is the dentin permeability of the drug. A high
concentration of drug is added to the first chamber and media in the
second chamber can be periodically replaced with drug-free media so that
the term (cB-cA) will approximate--cA.

[0040] Anatomic factors influence P since P∝1/L, where L is tubule
length. This can be standardized using dentin slices of the same
thickness. The diameter of the tubule is an important determinant of flux
since, P∝r2, the tubule radius raised to the second power. By
using teeth from young subjects, the value of this term can be maximized
since the tubules narrow as people age. By controlling for these anatomic
factors it is possible to define the conductance of the dentin to the
particular drug. Electrostatic charge and solubility influence
conductance. Since the tubule diameter is quite large compared to
molecular dimensions, molecular weight has little effect on transdentinal
diffusion over a range of sizes including those of most anti-inflammatory
drugs. The large molecular size and other characteristics of lipids-based
agents may have a limiting effect on their transdentinal flux.

[0041] Using the method of this invention, agents that exhibit potent
inhibition of inflammatory responses and cross the dentin barrier (i.e.,
a high P value) can be identified. Agents that efficiently diffuse
through the dentin can be used in combination with the delivery system
described herein to provide patients with more comfortable, cost
effective and reliable dental care, e.g., following invasive dental
processes or dental caries.

[0042] The invention is described in greater detail by the following
non-limiting examples.

Example 1

Materials and Methods

[0043] Dentin Specimen Preparation. Caries-free human third molars from
subjects less than 30 years of age were used in this study. Teeth were
collected in 1% phenol then debrided of adherent hard and soft tissue and
externally sterilized by soaking in chlorine bleach for 2 minutes
followed by 2% hydrogen peroxide for 2 minutes. Since the pulp space was
not sterilized, the teeth were handled with infection control precautions
throughout the experimental procedure. Teeth were used within one month
of collection.

[0044] The teeth were sectioned perpendicular to their long axis, using a
low speed saw (ISOMET, Buehler Ltd., Lake Bluff, Ill.) with a diamond
blade and deionized water lubricant. One-half millimeter thick sections,
free of occlusal enamel, were obtained. Since there was particular
interest in deep dentin in this study, some sections were partially
perforated by pulp horns. A grove was made on the pulpal side of each
disk using an abrasive disk on a low speed dental handpiece (MTI
Precision Products, Lakewood, N.J.) in order to facilitate later
fracturing. For disks that were examined with the SEM, the occlusal side
was polished with a series of abrasives to remove striations left behind
by the diamond blade (all polishing supplies from Buehler). Initially,
600 grit silicon carbide paper was used with water lubrication, followed
by an aqueous slurry of 3 μm diamond paste on a felt pad and finally
an aqueous slurry of 0.25 μm diamond paste on a felt pad. The polished
disks were sonicated for 2 minutes (model 450 SONIFIER, Branson, Danbury,
Conn.) at 30% power setting and 50% duty cycle. The disks were then
etched for 2 minutes in 0.5 M EDTA at pH 7.4 with agitation. The disks
were rinsed in 0.9% NaCl and stored in 0.9% NaCl until use. Immediately
prior to the application of dye or other experimental agents, the disks
were fractured in half along the grove prepared on the pulpal side of the
section. All experimental treatments were applied to the occlusal surface
of the dentin specimen.

[0045] Dye Staining.

[0046] Nine tooth slices were etched with EDTA and fractured in half as
described above. Immediately prior to use, the disks were rinsed in
deionized water. The color of the untreated dentin specimens were
examined with a tristimulus color analyzer (model CR221 Chroma meter,
Minolta, Osaka, Japan). This device measures three dimensions of a
specimens color: the brightness, as well as two aspects of chromaticity,
position on an axis representing red-green and position on an axis
representing yellow-blue. Since in these experiments light-yellow dentin
was stained with a dark-blue dye, the brightness values and position on
the yellow (positive numbers)-blue (negative number) axis were judged to
be the relevant color parameters and recorded for analysis. These chroma
meter readings were taken from three positions on each dentin surface
using a 3 mm diameter-measuring tip and specimen holder. One half of each
disk was then soaked for 60 seconds in a 33 weight % of the cationic
polymer polyquaternium-6 in 67 weight % of water, while the other half
was soaked in deionized water. Both disk halves were then rinsed with a
gentle stream of deionized water for 20 seconds, then placed into
separate vials containing a 0.5% solution of toluidine blue-0 (molecular
weight=305.83) (Harleco, Philadelphia, Pa.) for 1 minute. The polymer and
water-treated disk halves were then rinsed with deionized water for 1
minute, blotted dry and reexamined, under identical light conditions,
with the color analyzer with three readings taken from each disk half.

[0047] Three other dentin specimens were prepared and treated as described
above. Following toluidine blue staining, the two halves of each dentin
disk were photographed (DP12 Microscope Digital Camera System, Olympus,
Tokyo, Japan) side by side (occlusal surface up) in order to visually
compare the intensity of staining between the polymer and water treated
halves of the tooth slice.

[0048] Latex Beads and Scanning Electron Microscopy (SEN).

[0049] Polished, EDTA-etched dentin disks were fractured in half and
rinsed in deionized water prior to use. As with the dye staining
experiments, one half of each disk was soaked in the cationic polymer
solution for one minute while the other half was exposed to deionized
water. Both disk halves were rinsed in deionized water for 20 seconds.
The two disk halves were then soaked in a 4 weight % percent dispersion
of either anionic or cationic latex beads in water for one minute in
separate bottles. The disk halves were then gently rinsed with deionized
water for 20 seconds and allowed to air-dry for at least 24 hours prior
to preparation for SEM analysis. The two dentin disk halves from each
disk (one polymer treated before bead application the other water
treated) were attached to aluminum SEM sample mounts with silver paint
(Ted Pella Industries, Reading, Calif.), then sputter coated with gold
using an SEM Coating System (BIO-RAD, Hercules, Calif.), and examined
using a S-2500 Scanning Electron Microscope (Hitachi, Pleasanton,
Calif.). The split dentin disks were examined under low magnification
(35× magnification) and areas that were near the center of the disk
and symmetrically situated on either side of the fracture were selected
for higher magnification examination and photomicrography. In this way,
the effects of cationic polymer application on bead adhesion could be
examined in areas of equivalent tubule morphology (Ahmed, et al. (2005)
J. Oral Rehabil. 32:589-97). This procedure was conducted on 11 tooth
slice pairs, five for the cationic and six for the anionic beads,
respectively. The number of beads in a representative high power (6000
magnification) image from each of 22 disk halves examined was counted by
an investigator who did not know what treatment was applied, with the aid
of the particle counter tool in the public domain program, NIH Image. In
this study the enamel edges of the tooth slices were used to handle the
specimens, hence the ability of the beads to adhere to enamel was not
examined.

[0050] Experimental Treatments.

[0051] The cationic polymer used in this study was a 33 weight % water
solution of (poly) 2-propen-1-aminium, N,N-dimethyl-N2-propenyl
chloride, CAS No. 26062-79-3 (MERQUAT 106 Nalco Company, Naperville,
Ill.). The polymer is composed of repeating cationic quaternary nitrogen
groups with the following unit structure.

##STR00001##

[0052] This polymer was selected for these experiments because of its high
cationic charge density. This type of polymer has affinity for hair and
is used in commercial products under the designation polyquaternium-6.
The 33 weight % in 67 weight % water solution used in these experiments
had a low viscosity and was readily washed off the dentin surface.

[0053] The two types of latex polymer beads used in these experiments were
manufactured by Interfacial Dynamics (Eugene, Oreg.). Both had a particle
diameter of approximately 0.5 μm. Cationic beads had amidine surface
groups and anionic beads had carboxylic acid groups. The anionic and
cationic beads used had equivalent charge densities on their surfaces.
Both the amidine and carboxylate beads were used as 4 weight %
surfactant-free aqueous dispersions, in which the charge on the
individual particles stabilized the emulsion.

[0054] Data Analysis.

[0055] Chroma meter readings and bead counts were determined as
mean±standard deviation. A one way analysis of variance (ANOVA) with a
pair-wise Tukey-Kramer test was performed using the JMP statistical
program (SAS Institute Inc., Cary, N.C.) in order to determine if
significant differences existed in brightness values and yellow-blue
values between unstained dentin, dentin that was treated with polymer
prior to toluidine blue staining and stained dentin that was not polymer
treated. The same statistical test was used to determine if there was a
significant difference in bead counts per high power field between
water-treated dentin exposed to the cationic beads, polymer-treated
dentin exposed to the cationic beads, water-treated dentin exposed to the
anionic beads, and polymer-treated dentin exposed to the anionic beads.
Significance was set at the p<0.05 level.

Example 2

Dye Staining

[0056] Prior to staining, the right half of a tooth slice was treated with
the cationic polymer solution and the left half was treated with
deionized water. The water-treated part of the slice showed intense blue
staining of the dentin and light enamel staining. The stained dentin was
the same color as the dye solution indicating orthochromatic toluidine
blue staining. In initial studies, it was observed that dentin from deep
sections stained more intensely than shallow dentin and the dentin that
was occlusal to the pulp horns stained more intensely than dentin under
the occlusal fissures. On the half of the slice treated with the cationic
polymer, faint blue staining could be seen particularly close to the pulp
horns. Overall, the cationic polymer-treated enamel was largely
unstained. In two other sets of split dentin disks, the halves treated
with cationic polymer were stained much less intensely than the
water-treated halves.

[0057] Chroma meter readings obtained from dentin prior to stain
application and from stained dentin with and without polymer application
were obtained. Unstained dentin had a high chroma meter lightness value
of 86.4±4.7. Water-treated dentin had a significantly (p<0.05)
reduced lightness value of 20.1±5.5 when stained with toluidine blue.
In contrast, dentin that was treated with the cationic polymer prior to
dye staining had a brightness value of 83.2±5.5; however, this value
was not significantly (p>0.05) different from the value for unstained
dentin, but was significantly (p<0.05) higher than the value for
dentin that did not receive the cationic polymer treatment prior to
staining. Unstained dentin had a chroma value on the yellow-blue scale of
16.1±5.8 indicating a pale yellow color. Water-treated dentin that was
stained with toluidine blue had a significantly (p<0.05) different
chroma value of -27.4±3.0 indicating a color shift to blue. Dentin
that was cationic polymer treated prior to dye staining had a chroma
reading of 8.4±7.7, this was significantly (p<0.05) different from
both the chroma value for unstained dentin and the value for dentin that
was stained without cationic polymer treatment indicating a small but
detectable color shift from yellow to blue.

Example 3

Cationic Beads

[0058] SEM observations of polished, EDTA-etched dentin washed with water
and then treated with a dispersion of 0.52 μm diameter cationic beads
revealed a surface heavily covered with beads. Beads could be seen
covering the intertubular dentin, the orifices of some of the dentinal
tubules and adhering to the tubule walls below the surface. In contrast,
dentin in the half of the slice treated with the cationic polymer prior
to the application of the beads showed few beads on the dentin surface or
in the tubules. The heavy coating of the dentin with beads on the half of
the slice exposed to water prior to the cationic beads and the lack of
bead attachment in slice halves treated with cationic polymer solution
was seen in the four other slices examined in the SEM. Particle counts
obtained from water-treated and polymer-treated dentin surfaces that were
then exposed to the cationic beads indicated that polymer treatment
significantly reduced the number of beads adhering to the dentin surface.
Quantitatively, 649±334.1 cationic beads per high power surface
adhered to water treated dentin as opposed to only 9.8±4.3 beads per
high power field in the disk halves that were treated with the cationic
polymer prior to bead application. This difference was statistically
significant (p<0.05). Examination of the polymer-treated dentin by
SEM, revealed an appearance typical of etched dentin, as the cationic
polymer coating could not be observed under the SEM.

Example 4

Anionic Beads

[0059] Polished, EDTA-etched dentin treated first with water, then with a
dispersion of 0.45 μm diameter anionic beads were observed under the
SEM to be uniformly coated with beads. While few beads were observed over
the tubule orifices, beads were observed to line the walls of some of the
tubules. In an additional five specimens exposed to water prior to
treatment with the anionic beads, the same pattern of bead attachment was
observed; a uniform coating of the dentin surface with beads. Treatment
of the other half of the same dentin slice with the cationic polymer
prior to application of the anionic beads changed the pattern of bead
attachment. Irregular clumps of beads interspersed with relatively bare
areas were seen to cover the dentin surface. Some of the dentin surfaces
treated with the cationic polymer were observed to be relatively free of
beads. Other specimens had areas of high bead density interspersed with
relatively bear areas. Overall cationic polymer treatment significantly
(p<0.05) reduced the number of anionic beads seen to adhere to the
dentin surface with 1012±163.63 anionic beads per high power field
adhering to water-treated dentin verse 411±323.36 beads adhering to
cationic polymer-treated dentin. The number of anionic beads adhering to
non-polymer-treated dentin surfaces did not differ significantly from the
number of cationic beads adhering to untreated dentin surfaces
(p>0.05).

Example 5

Silica Particle Adhesion to Dentin and Coverage of Tubule Orifices

[0060] To further examine the use of porous or hollow beads in a dentinal
drug delivery system, uniform 0.5 μm silica particles were examined
using SEM. Dentin disks were polished and etched using standard methods.
Three types of silica particles were examined: amino (--NH2) beads
having a weak positive charge at physiological pH; hydroxyl (--OH) beads,
which would have a neutral charge; and acid (--COOH) beads that are
strongly negatively charged. Two and five percent dispersions of the
beads were prepared by high power sonication. The dispersions were
applied to moist dentin surfaces with a foam brush for 1 minute. The
specimens were then exposed to deionized water, allowed to dry and
prepared for SEM observation. Two thousand power images were prepared for
analysis. Three dentin specimens were treated with each suspension. In
this study, the image analysis software, Image J, was used to examine
tubule coverage by the bead dispersions.

[0061] Dentinal tubules were evident in this analysis. For dentin surfaces
treated with 2% acid (--COOH) beads, the surface was covered with
particles; however the tubules were relatively open. Previous
investigations observed a similar pattern of dentin coverage with --COOH
functional latex beads. For dentin surfaces treated with 2% --NH2
beads, in addition to dense coverage of the surface the dentin, the
tubules were covered. Similar to the amine functional beads, dentin
treated with --OH bearing silica provided a dense coverage of the surface
as well as beads occupying the orifices of the dentinal tubules.
Treatment of the dentin with 5% suspensions of the beads yielded similar
patterns of dentin and tubule coverage.

[0062] Using Image J, the open tubule percent of each dentin surface was
calculated. Since images had to be converted into a binary format the
tubule size may have been underestimated. FIG. 1 shows the percent open
tubule (6 observations each group) for control (untreated), COOH--,
NH2-- and OH-treated dentin. COOH bead treated and untreated dentin
surfaces had the same proportion of the surface as open tubule. In
contrast NH2 and OH bead treated dentin had very small amounts of
their surface as open tubule.

[0063] These results indicate that a variety of hydrophilic particles
attach to dentin and resist water washing. Although the --COOH beads
attached to the dentin surface, these beads were incapable of bridging
over the tubule orifice in the same fashion as the other two bead types.
Since the absolute magnitude of the surface charge is higher for --COOH
than for NH2, electrostatic repulsion may hinder the formation of
these bridging structures.

[0064] These results indicate that positive or neutral beads will be of
use as drug carriers to deliver drugs to dentin.

Example 6

Screen for Anti-Inflammatory Agents

[0065] The direct effect of drugs and lipids on inflammatory mediator
production in RAW cells was determined. This analysis indicated that,
compared to oleic acid, both aspirin and ibuprofen were effective
inhibitors of LPS-evoked PGE2 production. Notably, the basal levels of
PGE2 production were low in the absence of LPS stimulation (less than
1000 pg/ml). Similarly, aspirin effectively inhibited LPS-evoked NO
production. DHA also inhibited NO production but to a lesser extent than
aspirin. Measurable amounts of NO were produced in the absence of LPS
stimulation (˜15 μM). Both aspirin and DHA appeared to cause a
small (not statistically significant) reduction in this spontaneous NO
production. This analysis indicated that 5 mM aspirin significantly
reduced NO production, whereas 0.5 mM and lower concentrations (0.05,
0.005, and 0.0005 mM) did not significantly reduce LPS-evoked NO
production. Although both aspirin and DHA treatment reduced NO
production, combined treatment produced no evidence of a synergistic
effect. In contrast to aspirin, ibuprofen had no effect on LPS-evoked NO
production, even at a concentration of 50 μM, which inhibited
LPS-evoked PGE2 production. Levels of aspirin used in this study appeared
to be non-toxic to cells.

[0066] Transdentinal diffusion of aspirin and ibuprofen was elucidated
using the screening method of this invention. Diffusion experiments were
performed with aspirin or ibuprofen solutions in the first chamber (the
donor compartment). Following 24 hours of diffusion, fluid was withdrawn
from the second chamber and applied to RAW cells. When these RAW cells
where stimulated with LPS, PGE2 production was significantly reduced by
the diffusate of both the aspirin and ibuprofen experiments. In aspirin
diffusion experiments LPS-evoked NO production was also depressed,
whereas ibuprofen diffusion experiments did not attenuate LPS-stimulated
NO production.

[0067] These experiments examined the ability of drugs to suppress
inflammatory pathways evoked by bacterial endotoxin challenge. LPS
injection induces severe local and systemic inflammatory responses.
Aspirin demonstrated unexpected utility as it inhibited both PGE2 and NO
production and diffused through dentin in sufficient quantities to
suppress these important inflammatory pathways. Like aspirin, ibuprofen
also diffused though dentin and inhibited PGE2 production. However,
ibuprofen did suppress NO production. DHA, a ω-3 fatty acid, also
inhibited LPS-provoked NO production but to a lesser extent than aspirin.
This property was not shared by oleic acid a monounsaturated fatty acid.
These results highlight the utility of ω-3 fatty acids in
modulating the inflammatory process without suppressing it to the same
extent as NSAID agents.

Patent applications by Kenneth Markowitz, Fanwood, NJ US

Patent applications by UNIVERSITY OF MEDICINE AND DENTISTRY OF NEW JERSEY